Custom cell culture environments offer great promise. Cell cultures and cell co-cultures may allow scientists to discover cell behavior and to safely determine whether a proposed therapeutic agent may be effective as well as safe for treating a particular disease. Recent advances in micropattern cell culture development have proven invaluable in increasing our understanding of the structure-function relationships of multi-cellular communities. Such micropattern cell cultures yield more control over cell-cell interaction, particularly when precise and reliable cell spacing is achieved. The study of tissue organization at the micro-environment level has revealed insights into areas as diverse as angiogenesis, hepatocyte function, calcification of bone derived cells and neuronal growth cone guidance. Additionally, micropattern cell cultures provide researchers with the ability to scale their experiments and thereby more effectively and efficiently conduct tests and experiments in parallel. This increases the rate at which scientific discovery can occur and speeds the development of new drugs and more quickly elucidates the mechanisms of certain diseases.
However, forming micron level cellular islands can be a difficult process as it requires tools that can precisely manipulate small quantities of cells, such that cells are spaced close enough to be measured in microns. Additionally, cells and other biological material that are needed to build the cellular islands are delicate and readily perishable, thereby requiring manipulation techniques that are not caustic, or otherwise harmful to the biological materials being manipulated.
Numerous techniques have been investigated for forming cellular islands on a substrate. These techniques include the chemical modification by photolithography of a glass substrate, and jet printing techniques that use small, low temperature printing heads to dispense drops of liquid that carry cellular material to the substrate. One such printing technology includes the Celljet cell printer manufactured and sold by the Digilab Company of Holliston, Mass. The Celljet printer prints cells as part of a liquid dispensing operation that dispenses a droplet of fluid containing cells. The droplet may be dispensed onto a microtiter plate or to a multi-plate. Other techniques for forming cellular islands include micro-contact printing and laser directed cell writing. Direct micro-contact printing typically involves the use of a structured, inexpensive, elastomeric stamps usually made of polydimethylsiloxane (PDMS) which have a relief pattern at the micron scale. These stamps usually allow the parallel deposition of molecules onto the target substrate surface. During contact, materials from the PDMS stamp are transferred onto the substrate. This transfer requires an efficient and usually quick transfer of molecules from the stamp surface to the substrate. One such technique is described in Direct Micro-contact Printing of Oligonucleotides for Biochip Applications, Thibault, et al. Journal of Neurobio Technology (2005) 3:7. As described in this reference, an electrobeam lithography approach, was used to etch a silicone master mold into which liquid PDMS may be poured. The liquid PDMS may be degassed, and then cured thermally. The PDMS may then be removed from the master to provide a stamp which can be used multiple times, depending upon the surface chemistries. The micron features of the stamp contact a substrate and prints a material onto the substrate at the point of contact. The success of the process depends in part on the successful contact of the elastomeric stamp with the substrate.
The physical contact between the micron features of the elastomeric stamp and the substrate must be made precisely and consistently across the stamp or the elastomeric material will fail to act as a proper mask during the etch process or fail to print the desired material onto the substrate. Sometimes it is the case that only partial contact is made and a result is a “coffee ring” pattern that correctly forms a perimeter of the pattern but fails to make successful contact for the interior portion of that pattern. Failure to form correct patterns means that the testing or experiment protocol cannot be followed and this can prevent the patterned substrate from being used within the experiments.
Accordingly, there is a need in the art for systems and methods that provide improved micro-contact stamps and stamp processes.